Anaerobic microorganisms are organisms which have the ability to grow in the absence of oxygen. The term, anaerobe, is commonly reserved for those organisms which, in nature, normally grow in (or, in the case of obligatory or strict anaerobes, can only grow in) anaerobic habitats. These are environments in which oxygen is absent. The anaerobes lack the ability to synthesize an oxygen-linked respiratory chain and must, therefore utilize alternate energy-yielding metabolism processes, such as fermentation, photosynthesis, etc.
Some anaerobic-like microorganisms can also grow under aerobic conditions. These are called facultative anaerobes or microbes as opposed to strict or obligatory anaerobes. In essence, these organisms generally prefer an oxygen environment but are capable of living and growing in its absence. Consequently, facultative microbes are capable of growing and/or surviving in the presence or absence of molecular oxygen.
Anaerobe microorganisms are economically important in health care, in food industry, and in the environment. However, anaerobes are difficult to isolate and grow because of their requirement for anaerobiosis and a chemically reduced state. To achieve these conditions requires specially prepared media to produce the reduced state and devices to create and maintain an oxygen free environment. The techniques that are required to use these devices add time and cost to working with anaerobes. New approaches, such as use of biocatalytic oxygen reducing agents, see for example the Oxyrase® microbiological products and processes, (U.S. Pat. Nos. 4,476,224; 4,996,073; 5,240,853; 5,432,083; 5,362,501; and 5,482,860) and specially design Petri dishes or culture plates, such as OxyDish™, (U.S. Pat. Nos. 5,830,746 and 5,955,344) of Oxyrase, Inc., Mansfield, Ohio (the assignee of the present invention), have simplified and reduced costs for isolating and growing these important microbes. The above mentioned patents are included herein by reference.
However, the very nature of anaerobes and the places that they are found conspire to make their isolation difficult. Because anaerobes lack respiratory enzymes for utilizing oxygen, they derive less energy from substrates than do facultative microbes, that have these enzymes. That means when anaerobes grow together with facultative microbes, some facultative microbes grow faster and more efficiently than do the anaerobes.
Furthermore, the majority of clinical specimens (80% to 85% ) are mixed with both anaerobe and facultative microbes. Samples taken from food and from the environment often contain mixed cultures. If grown together, the facultative microbes often out grow the anaerobes, resulting in a diminution of the anaerobe numbers relative to the facultative microbes.
Additionally, when a specimen is spread onto a plate for isolated colonies, often the number of individual colonies obtained is about 100 or less. If the ratio of anaerobe to facultative falls to about 1%, the isolation of the anaerobe on a plate may be missed.
The crowding of facultative microbes and the presence of a relatively infrequent anaerobe on the same plate makes isolation of the anaerobe in pure form difficult. Not only is the anaerobe colony difficult to find, but it is often located in a crowded field that makes picking the colony difficult to do.
To be able to efficiently isolate a pathogenic anaerobe from among a mixed flora in a clinical specimen is important to the patient and can impact the cost of health care associated with treating that patient. The time it takes to recognize the presence of an anaerobe in a food sample is important to public health issues and to the economics of food packaging, storage, and shipment.
The object of this invention is to provide a medium (or a medium composition), process and/or device that retards or restricts the growth of facultative microbes, but not that of anaerobe microbes. The medium composition comprises a nutrient medium, an oxygen reducing agent (preferably, biocatalytic) and a cyanide, azide, and/or other related inhibitor compounds. These compounds act by chemically, irreversibly bonding to key enzymes or factors in the respiratory chain, thereby disabling them from handling oxygen. Such selective use of inhibitors restricts the growth of facultative microbes and removes the relative advantage that facultative microbes have when competing with anaerobe microbes.
Accordingly, it is an object of the present invention to provide an improved media composition, method and/or device for isolating and/or identifying anaerobic microorganisms contained in a mixed culture sample with facultative microorganisms.
These and other objects and features of the invention will be apparent from the following summary and description of the invention and from the claims.